The distribution of fitness effects (DFE), or beneficial mutation rate spectrum, is the rate at which mutations of each fitness effect occur in a particular genotype. We have previously characterized the DFE in an initially isogenic yeast population using high-resolution lineage tracking and shown that it can be used to predict the evolutionary dynamics of large populations in the lab. In more complex evolutionary scenarios where initial genotypes are heterogeneous, the evolutionary dynamics depends on both the fitness and the DFE of each genotype. Yet, little is known about how the DFE varies across genotype space. Here, we focus on measuring how the DFE depends on the genotype using a novel tandem integration double barcoding system. This system relies on two incompatible loxP variant sites in the genome, which sequentially bring two barcoded plasmid libraries (BC1 and BC2) to the common genomic location. The trajectory of each double barcode lineage can subsequently be monitored by pooled growth and amplicon sequencing. We have generated a large haploid segregant pool from a cross between genetically divergent strains, the lab strain BY4741 and the pathogenic clinical isolate YJM789. We have picked and verified ~200 segregants, each barcoded with a unique BC1, and measured their fitness by pooled competition to find a subset that represent a broad spectrum of initial fitnesses. To measure the DFE of many segregants, we are barcoding each with ~100,000 BC2 barcodes and generating ~5 pools that contain overlapping segregants. The construction of segregant pools are guided by numerical simulations to maximize the number of segregants for which the DFE can be estimated. Serial batch evolution with these pools in three different environments will be used to examine how much the DFE varies between segregants, and if and how much the DFE depends on the initial fitness. Next, we will sequence the genome of each segregant to perform a quantitative trait loci (QTL) analysis for various features of the DFE. This QTL study will test the hypothesis that some genetic loci explain some variance in the DFE and thereby modulate evolvability.